Urinary miR-16-5p can be used as a potential marker of endocapillary hypercellularity in IgA nephropathy

The most prevalent primary glomerulonephritis and leading cause of end-stage renal disease worldwide is IgA nephropathy (IgAN). More and more studies are describing urinary microRNA (miRNA) as a non-invasive marker for a variety of renal diseases. We screened candidate miRNAs based on data from three published IgAN urinary sediment miRNAs chips. In separate confirmation and validation cohorts, we included 174 IgAN patients, 100 patients with other nephropathies as disease controls (DC), and 97 normal controls (NC) for quantitative real-time PCR. A total of three candidate miRNAs, miR-16-5p, Let-7g-5p, miR-15a-5p were obtained. In both the confirmation and validation cohorts, these miRNAs levels were considerably higher in the IgAN than in NC, with miR-16-5p significantly higher than in DC. The area under the ROC curve for urinary miR-16-5p levels was 0.73. Correlation analysis suggested that miR-16-5p was positively correlated with endocapillary hypercellularity (r = 0.164 p = 0.031). When miR-16-5p was combined with eGFR, proteinuria and C4, the AUC value for predicting endocapillary hypercellularity was 0.726. By following the renal function of patients with IgAN, the levels of miR-16-5p were noticeably higher in the IgAN progressors than in the non- progressors (p = 0.036). Urinary sediment miR-16-5p can be used as noninvasive biomarkers for the assessment of endocapillary hypercellularity and diagnosis of IgA nephropathy. Furthermore, urinary miR-16-5p may be predictors of renal progression.


Methods
Overall study design. In this research, we reviewed a total of three profiles of urinary sediment miRNAs in the literature on IgAN by Zhi-Yu Duan, Cheuk-Chun Szeto, and Nannan Wang [4][5][6] , and found that miR-16-5p, Let-7g-5p, and miR-15a-5p were expressed in the same trend in the three miRNAs profiles, all of them were significantly higher in IgAN compared with normal controls, thus identifying the target miRNAs for this study, as shown in Fig. 1 Sample preparation. Collect 50-100 ml of urine sample from the patient early in the morning on the day of kidney biopsy, place it in a 4 °C refrigerator for temporary storage after collection, and process it within 6 used. According to the situation, data were presented as mean ± standard deviation or median (inter-quartile range). The 2 (−ΔΔCt) relative quantification method was used to compare the expression of miRNAs. Shapiro-Wilk tests were performed on normality analysis. For normally distributed data, the t test or one-way ANOVA were employed. For data which weren't normally distributed, the Mann-Whitney or Kruskal-Wallis rank test was applied. Correlation analysis was performed using the Pearson or Spearman methods to explore the association between miRNAs and clinical and pathological indicators. The sensitivity and specificity of miRNAs for the diagnosis of IgA nephropathy were calculated utilizing receiver operating characteristic (ROC) curves and area under the ROC curves (AUC). P < 0.05 was used to define statistical significance for the results. All computations were performed with two-tailed probabilities.
Ethical approval. The studies involving human participants were reviewed and approved by Research Ethics Committee of Chinese PLA General Hospital (approval number S2018-206-01). The patients/participants provided their written informed consent to participate in this study.  Table 1. The expression trends of miR-16-5p, Let-7g-5p, and miR-15a-5p were found to be consistent in the three expression profiles, and all were noticeably higher in IgAN than in normal controls with at least a 10 fold-change, according to Table 2.

Results
Clinical and pathological features of patients with IgA nephropathy. We included a total of 174 patients with IgAN, 100 disease control patients and 97 healthy individuals. The clinical and demographic details of the study participants are summarized in Table 3. The pathological features of the IgAN group are listed in Table 4.

Correlation between miR-16-5p and renal progression.
To confirm the efficacy of urine sediment miRNAs in predicting the development of renal function in IgAN, 122 IgAN patients were followed for a mean of 8.5 (4-18) months, with a total of 54 IgAN followed for >12 months, 14 of whom were progressors, including one patient who developed ESKD with eGFR ≤15 ml/min/1.73m 2 , and 40 patients in the non-progressors. The level of miR-16-5p was considerably greater in the progressor than in the non-progressor (P = 0.036) (Fig. 3B), suggesting that the higher the baseline miR-16-5p expression, the faster the decline in eGFR.

Discussion
In this study, we identified three miRNA targets that differed significantly in urine levels between IgA nephropathy and normal controls. Among them, urinary miR-16-5p is a potential marker of endocapillary hypercellularity in IgA nephropathy, which has a good diagnostic value and may be a predictor of renal function progression in IgA nephropathy. Previous research has looked into the differences in miRNA levels in the urine of people with IgAN 4-6 . For example, Wang et al. 6 reported that miR-3613-3p expression was significantly downregulated in the urine of IgAN patients and miR-3613-3p and miR-4668-5p correlated with disease severity. MiR-25-3p, miR-144-3p, and miR-486-5p in urinary sediment may be diagnostic markers of IgA nephropathy and are mainly derived from urinary red blood cells. Higher levels of miR-144-3p expression are linked to lower urine protein and better renal function 4 . Similarly, Liang et al. 11 reported urinary miR-21 and miR-205 as prospective prognostic markers for  www.nature.com/scientificreports/ the assessment of interstitial tubular damage in IgAN, and urinary miR-204 was reported as a diagnostic tool for IgA nephropathy 5 . Therefore, we reviewed the urinary sediment miRNA profile published in previous studies on IgAN and found that miR-16-5p, Let-7g-5p, and miR-15a-5p expression trends were consistent in three studies [4][5][6] , all of which were noticeably increased in patients with IgAN in contrast to normal controls, with a fold change > 10, thus identifying the target miRNAs for this study. The PCR results of the confirmation cohort showed significant differences between IgAN and normal patients, totally supporting the trend of the microarray study discussed above. We then included disease controls for non-IgAN in an independent validation cohort, and the results suggested that urinary miR-16-5p may be a diagnostic biomarker of IgAN. Analysis of the ROC curve revealed good specificity and sensitivity. In addition to the diagnostic value, we further investigated the clinical significance of these urinary sediment miRNAs. Proteinuria, eGFR and Oxford classification were all closely associated with the progression and prognosis of IgAN [12][13][14] . In our results, miR-16-5p levels were significantly higher in the urine sediment of patients with E1 in contrast to E0 patients, and blood complement C4 levels were lower in E1 compared with E0 patients, further confirming that endocapillary hypercellularity is a hallmark of active inflammation 15 and that endothelial cell proliferation is primarily the result of inflammatory cell infiltration 16 . Bao et al. 17 used mesangial cells to stimulate endothelial cells, and their miRNA microarray results showed high expression of miR-16-5p. Therefore, we hypothesized that under galactose-deficient IgA1 stimulation, mesangial cells secreted substances that stimulated endothelial cells to overexpress miR-16-5p. Studies have demonstrated that in patients with IgAN who are not receiving immunosuppressive therapy, endocapillary hypercellularity is a reliable indicator of the rate of renal function loss 18 . Thus the lack of prognostic value for the E score in other studies is likely the result of immunosuppression-related treatment bias 19,20 . A number of subsequent studies have provided evidence for immunosuppressive treatment of intracapillary proliferative IgAN 21,22 . In addition, Let-7g-5p was positively correlated with baseline proteinuria and negatively correlated with eGFR. The higher the expression level of Let-7g-5p, the more its 24-hour urinary protein and the worse its renal function, suggesting that Let-7g-5p may be associated with prognosis. miR-15a-5p was not associated with proteinuria, eGFR, or pathological indicators. The follow-up of renal function in IgAN patients revealed significant differences in miR-16-5p expression levels between the progressor and non-progressor, all of which suggest that urinary sediment miRNAs may be related to the progression and prognosis of IgAN.
Although the biological functions of miR-16-5p, Let-7g-5p, and miR-15a-5p have not been clarified, bioinformatics analysis may elucidate the roles of these miRNAs by predicting hub genes. A total of 133 common target genes of miR-16-5p, Let-7g-5p, and miR-15a-5p were obtained from database miRDB and starBase. By Gene Ontology (GO) enrichment analysis 23 , the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis 24,25 and the Protein-Protein Interaction (PPI) network construction 26,27 , the hub gene CCND1 may have a significant impact on how these miRNAs regulate the biological process of IgAN unpublished). According to GO analysis, the functions of these miRNAs are mainly enriched in the regulation of catabolic, metabolic and renal water homeostasis. Antidiuretic hormone (AVP) is important for water homeostasis, and it has been shown that copeptin, a reliable and stable alternative to circulating AVP, was found to correlate with disease severity and prognosis in IgAN patients by detecting copeptin 28 . According to KEGG pathway enrichment analysis, these miRNAs were significantly enriched in FoxO signaling pathway, mTOR signaling pathway, and Wnt signaling pathway. The FoxO signaling pathway has been shown to play an important role in diseases associated with altered B-cell proliferation and/or activation 29 , while up-and down-regulation of some B cells was indeed found by studies of peripheral blood cell subsets in IgAN 30 . Activation of the mTOR www.nature.com/scientificreports/ signaling pathway causes activation of downstream signaling proteins such as p70s6K, leading to extracellular matrix deposition in glomerular mesangial cells, renal tubular and collecting duct epithelial cells, renal fibroblast activation, and renal interstitial fibrosis. Rapamycin, as an inhibitor of the mTOR signaling pathway, can effectively inhibit glomerular mesangial cell proliferation and extracellular matrix secretion by early low-dose application, reduce IgA deposition, and protect renal function 31 . It has also been demonstrated that excessive Wnt signaling pathway activation contributes to the development of IgAN 32 . In addition, A critical gene that controls the cell cycle is CCND1, and turning on its expression can encourage cell growth. One study found that aberrant miR-320 expression in IgAN urine and kidney tissue promoted B cell proliferation and increased the expression of CCND1 33 . In a rat model of IgAN, autophagy is inhibited, while the mTOR/S6k1 pathway is activated and CCND1 expression is increased 34 , all of which provide directions for further studies. Our study has some shortcomings. First, all samples were from a single center and were Chinese, whereas the prevalence of IgAN and disease characteristics vary considerably among different countries and ethnic groups, and further validation is still needed. Second, in this study, the basic demographic and clinical information (e.g., age, renal function, or proteinuria) of patients with IgAN was not exactly matched to the diseased controls and normal controls, and a strict pairwise analysis was not performed due to differences in the number of subjects. In addition, the cellular origin of miRNAs in urine sediment is not clear. miR-16-5p, Let-7g-5p, and miR-15a-5p were reported to be the top 20 miRNAs in human blood erythrocytes 35 , so we speculate that these miRNAs may be derived from erythrocytes, but further experiments are still needed to verify them in urine. Finally, how miR-16-5p, Let-7g-5p, and miR-15a-5p act on IgAN via CCND1 remains to be further explored.
In conclusion, in the first part of the study, we reviewed and validated a significant rise in miR-16-5p, Let-7g-5p, and miR-15a-5p in IgAN. Then, we found a significant correlation between miR-16-5p and endocapillary hypercellularity. Finally, we evaluated the effect of miR-16-5p on the progression of IgAN renal function and made some bioinformatic speculations. These findings support the idea that miR-16-5p is a potential marker reflecting endocapillary hypercellularity and predicting IgAN progression.

Data availability
The raw date supporting the conclusions of this article will be made available by the authors, without undue reservation. Correspondence and requests for materials should be addressed to G.-Y.C.